Premature births are not uncommon in Germany where around seven percent of newborns are born before week 37 of pregnancy. The WHO estimates that the numbers of preterm births are growing due to the increasing age of mothers. Doctors caring for the tiny patients are faced with a dilemma because regular blood samples required for the clinical monitoring of important blood parameters cannot be taken due to the infants’ low body volume.
Regular measurement of plasma proteins, which could provide essential information about the function and developmental state of various organs, has previously been impossible. A BMBF-funded cooperative research project with the participation of Prof. Dr. Dieter Stoll’s research group at the Albstadt-Sigmaringen University of Applied Sciences might have found a solution to the problem: the researchers hope that the analysis of plasma proteins in a small number of blood droplets will provide them with the information they need.
“The low weight of preterm infants is one of the main causes of high infant mortality,” said Dieter Stoll, professor of bio-engineering at the Albstadt-Sigmaringen University of Applied Sciences. “Delayed organ development and poor nutritional status can also lead to serious disabilities and chronic illnesses.” The regular monitoring of a variety of plasma proteins would improve the treatment options. The number of so-called diagnostic marker proteins allows precise statements to be made about the various bodily functions such as clotting, immune response, nutrient transport and protease inhibition. However, the withdrawal of a few millilitres of blood, required by established diagnostic procedures, is a high health risk for premature infants. Newborn and preterm infant screening programmes rely specifically on mass spectrometry methods to screen infants for amino acid and fatty acid metabolism defects as they are capable of producing reliable diagnostic results from relatively small sample quantities.
However, such a method is not yet available for the analysis of plasma proteins. The cooperative project, consisting of the Department of Paediatric and Adolescent Medicine at the University of Lübeck, Albstadt-Sigmaringen University of Applied Sciences, the NMI Natural and Medical Sciences Institute at the University of Tübingen and the companies HB Technologies AG (Tübingen) and Protagen AG (Dortmund), is set to change this situation: the partners have the ambitious goal to identify 50 plasma proteins in a single droplet of capillary blood using mass spectrometry.
The mass spectrometric analyses that will be used by Stoll’s team of researchers have a decisive advantage over established immunological test systems in that they allow the multiplex measurement of biological molecules. “Mass spectrometry enables the direct quantification of different analytes in a single sample. Moreover, the technique works effectively with very small sample volumes,” said Stoll. In general, mass spectrometric analyses used to infer the existence/quantity of an unknown protein of interest in a sample are based on proteolytic peptides that arise from the enzymatic digestion of the proteins. In addition, the quantity of proteins of interest can be determined simultaneously from several different peptides, a procedure that increases the reliability of the diagnosis. In order to then be able to reliably determine the quantity of the unknown protein of interest, defined quantities of so-called stable isotope-labelled proteins are added to the blood droplet under analysis. These reference proteins have the same characteristics as the proteins of interest, but their peptides have a slightly higher mass. The proteolytic peptides can therefore be distinguished with mass spectrometers due to their mass difference. The comparison of the signal strengths of the reference peptides with those of blood protein-derived peptides enables the researchers to quantify directly the amount of blood protein.
As proteins of interest to the researchers are generally present in a complex mixture and varied abundance, the required peptides need to be enriched from billions of other peptides prior to mass spectrometric analysis. Clinical research uses antibodies for carrying out this process. However, the method used, which is known as immunoaffinity enrichment, is also associated with significant disadvantages: first, the development of antibodies is a highly time-consuming and cost-intensive undertaking, and second, antibodies are very expensive reagents.
The solution sought by the project partners has two key advantages over traditional procedures: first, the possibility of adding reference proteins immediately after sample collection, and second, the method’s higher sensitivity and specificity due to immunoaffinity enrichment with special antibodies that are being developed within the researching group of Dr. Oliver Pötz at the NMI. In contrast to conventional antibodies, which only detect and bind to a single molecule, the special antibodies used in the project are able to bind a variety of peptides. “Around 100 antibodies are sufficient to enrich 10,000 human proteins,” said Pötz. Pötz uses people’s names where each letter stands for an amino acid, to illustrate how the so-called TXP (Triple X Proteomics) antibodies (see diagram) work.
Classical antibodies are peptide specific and bind to antigen domains (epitopes) that are between five and seven amino acids long. Taking people’s names as example, an antibody that is able to bind to the sequence “FABIAN” does not bind to the sequences “CHRISTIAN” or “JULIAN”. It is specific for FABIAN. In contrast, TXP antibodies can immunoprecipitate groups of peptides which share a common short sequence at the N- or C-terminal end. The TXP epitopes are only three to four amino acids long, which means that they are not restricted to one particular peptide, but that they can bind equally strongly to all members of a group of peptides that share the same terminal amino acid sequence. An “IAN” antibody can therefore not only bind to the sequence “FABIAN”, but also to “FLORIAN” or “SEBASTIAN”. The only prerequisite is that the amino acids must occur at the terminal end of a peptide. The “IAN” antibody would not be able to bind to “IANNI” because the anti-IAN antibody only binds to “IAN” when the N is located at the very C terminus of the peptide. The use of TXP antibodies therefore enables each antibody to simultaneously bind to several peptides from different proteins. This reduces the costs, facilitates the parallel analysis of several proteins in blood samples and therefore represents a crucial step on the path towards the more efficient analysis of blood proteins. Each project partner is contributing in a different way to the research objective. Protagen AG, the project coordinator, is specifically focused on the research and development of biomarkers and is tasked with the production of stably isotope-labelled reference proteins; HB Technologies AG brings to the project its skills in the construction and control of laboratory devices used in the life sciences. The Department of Biochemistry at the NMI Natural and Medical Sciences Institute at the University of Tübingen in Reutlingen produces and characterises the TXP antibodies used in the consortium’s mass spectrometry assays. The medical side of the project, which involves the provision of patient samples and the clinical evaluation of the methods, is being carried out by the Department of Paediatric and Adolescent Medicine at the University of Lübeck.
The new method not only facilitates the screening of infants, the technology platform also has the potential to be used in clinical trials. “A sample amount of 5 - 10 millilitres will most likely be sufficient for determining several diagnostic parameters. This also makes the method highly attractive for clinical trials testing the safety and efficiency of medical products given to children,” said Stoll. Research projects based on TXP immunoaffinity enrichment from the researching group of Dr. Oliver Pötz are also being planned in other areas. For example, the detection of allergens in food would be one conceivable application. “In this area, colleagues of the Albstadt-Sigmaringen University of Applied Sciences have already established test systems on the basis of nucleic acids, which we would like to expand with our mass spectrometry technologies,” said Stoll.The project PlasmaQBaby is funded by the German Federal Ministry of Education and Research (BMBF) under its KMU-innovativ-8 (Innovative SMEs) high-tech strategy and managed by Jülich Project Management (PTJ).
Further information: Prof. Dr. Dieter StollAlbstadt-Sigmaringen University of Applied Sciences, Pharmaceutical Technology & Biomedical SciencesAnton-Günter-Straße 5172488 SigmaringenTel.: +49 (0)7571 -732-8525E-mail: stoll(at)hs-albsig.de